Gap junctions are specialized regions of cell-to-cell contact in which hexameric oligomers, called connexons, dock end-to-end across a narrow extracellular gap, allowing the intercellular exchange of nutrients, metabolites, ions and small molecules. Previously, this research program focused on the 43 kDa connexin (Cx43) channel, which mediates ionic conduction between cardiac myocytes, thereby regulating the normal heartbeat, but also mediating potentially fatal cardiac arrhythmias. We have now expanded our research to include Cx26, mutations in which are the predominant cause of inherited, nonsyndromic deafness, and Cx40, which forms high conductance channels in the specialized conducting tissue of the heart. For the next cycle, we will pursue 3 specific aims: (1) Our major aim, to which we will devote 70% effort, is to use X-ray crystallography to determine an atomic resolution structure of one or more hexameric connexons, which includes improving upon the resolution of a recent 3.5 E structure of Cx26 (Maeda et al., 2009), as well as testing our cryoEM-based C1 model for the assignment and packing of the transmembrane ?-helices. (2) Our second aim, to which we will devote 20% effort, is to use electron cryocrystallography of 2D crystalline gap junction plaques isolated from cells to examine the higher resolution structure of the extracellular loops, in order to understand the molecular basis for isoform selectivity and how the packing of the loops forms a tight molecular seal to exclude the extracellular environment. (3) A third minor aim, to which we will devote 10% effort, is to use cryoEM and single particle image analysis of connexons to explore gating and regulation of a number of Cx26 truncation mutants. Of particular interest is the recent discovery that the N-tail of Cx26 forms a central gating plug (Oshima et al., 2007). We will perform a structure/function analysis by correlating the structure of truncation mutants with functional studies of channels incorporated into liposomes. Our research is enriched by several key collaborations: Dr. Andrew Harris uses a liposome assay to test the function of our Cx mutants; Dr. Ray Stevens is an expert membrane protein X-ray crystallographer; Dr. Qinghai Zhang has synthesized custom detergents for channel stabilization to enable 3D crystallization trials; and Dr. Anchi Chang is an expert in electron cryocrystallography. By this integrated approach, we will continue our quest to visualize an atomic structure of a gap junction channel.